Polymeric materials containing amino groups such as, for example, proteins and solid support-based organic and inorganic matrices (e.g. "aminopropyl glass") are often used as carriers of various materials including bioactive materials. Several methods are known for coupling carboxyl group-containing materials to such carriers.
The prior art has used coupling procedures to covalently link carboxylic group-containing biological macromolecules that are protective antigens of pathogenic bacteria. Acidic polysaccharides have been coupled to carrier proteins in order to form conjugate vaccines for increasing the immunogenicities of such polysaccharides. Another application is the coupling of acidic materials to solid carriers containing an amino groups for use in affinity chromatography. Such water-insoluble chromatographic media can be used, for example, for the isolation of antibodies from physiological fluids.
Another prior art method involves the activation of the carboxylic acid groups by hydroxysuccinimide derivatives. This method suffers from the same disadvantages as the carbodiimide procedure discussed, hereinabove.
A further known procedure employs adipic acid dihydrazide as a homobifunctional spacer. In the adipic acid dihydrazide method, the carboxylic acid group of a carbohydrate or polysaccharide is coupled to adipic acid dihydrazide in the presence of a water-soluble carbodiimide by way of the hydrazide linkage. The spacer is terminated by a strongly nucleophilic hydrazino group which can be coupled to the carboxylic acid group of a protein.
Potential disadvantages of this method include: (i) inter- and intramolecular crosslinking of the carboxylic acids group-containing carbohydrate or polysaccharide; and (ii) cross linking of the protein through its amino and carboxylic acid groups in the second phase of the conjugation.
U.S. Pat. No. 3,947,352 to Cuatercasas et al. discloses the periodate oxidation of polysaccharides and reaction of the aldehyde derivatives with polyhydrazide derivatives. The product of this reaction can then be coupled to various biologically active molecules.
Linkers or spacer compounds are also described by Fattom et al. Immunity, February 1992, pp. 584-589. The specific linkers disclosed by Fattom et al. are adipic acid dihydrazide and N-succinimidyl-3-(2-pyridyldithio) propionate. This article relates to the formation of conjugates of Staphylococcus aureus Type 8 capsular polysaccharide. The bond formed between these linkers and the capsular polysaccharide are identical. However, the N-hydroxysuccinimyl moiety reacts mostly with lysine amine groups of the protein while the hydrazide binds to the carboxyl in the protein.
U.S. Pat. No. 4,882,226 to Schutyser et al. relates to a carrier material which comprises a copolymeric core material which is covalently bonded to a hydrophilic coating material. The carboxyl groups on the core material are reacted with glycidol or glycidol derivatives and the hydrophilic material is linked directly to the core material or via various spacer groups.
U.S. Pat. No. 4,356,170 to Jennings et al. and U.S. Pat. No. 4,446,275 to Filka et al. teach procedures in which linking of polysaccharides is attained via the formation of an aldehyde moiety by periodate oxidation followed by coupling by reductive amination. This coupling is a direct linkage of the polysaccharide and the protein and would suffer from some of the same disadvantages as noted for the carbodiimide method, hereinabove. Additionally, this procedure requires the presence of vicinal hydroxy groups. Hence, oligosaccharide or polysaccharides which do not have vicinal hydroxyl groups cannot be coupled using this procedure. Care must be taken in order to prevent cross-linking during conjugate formation causing loss in conjugate solubility and activity.
The most successful of all carbohydrate pharmaceuticals so far have been the carbohydrate based, antibacterial vaccines 1!. The basis of using carbohydrates as vaccine components is that the capsular polysaccharides and the O-specific polysaccharides on the surface of pathogenic bacteria are both protective antigens and essential virulence factors. The first saccharide-based vaccines contained capsular polysaccharides of Pneumococci: in the United States a 14-valent vaccine was licensed in 1978 followed by a 23-valent vaccine in 1983. Other capsular polysaccharides licensed for human use include a tetravalent meningococcal vaccine and the Vi polysaccharide of Salmonella typhi for typhoid fever. The inability of most polysaccharides to elicit protective levels of anti-carbohydrate antibodies in infants and adults with weakened immune systems could be overcome by their covalent attachment to proteins that conferred T-cell dependent properties 2!. This principle led to the construction of vaccines against Haemophilus influenzae b (Hib) 3! and in countries where these vaccines are routinely used, meningitis and other diseases caused by Hib have been virtually eliminated. 4! Extension of the conjugate technology to the O-specific polysaccharides of Gram-negative bacteria provided a new generation of glycoconjugate vaccines that are undergoing various phases of clinical trials 5!.
Chemical synthesis may provide fragments of natural polymers that have the necessary geometry to mimic conformational determinants of the native polymer which may be useful in diagnostics or as components of vaccines. The synthesis of di- to penta-, tetra-, hexa-, octa-, and dodeca-saccharide fragments of the O-specific polysaccharide of Shigella dysenteriae type 1 6, 7! and the use of such synthetic oligosaccharides to map the carbohydrate binding specificity of anti O-specific polysaccharide specific murine monoclonal antibodies 8! have been previously reported. More recently, the synthesis of a hexadecasaccharide of Shigella dysenteriae type 1, consisting of consecutive tetrasaccharide repeating units, has been reported 9!.
U.S. Pat. No. 4,137,401 to Lemieux et al. (1979) describes carbohydrate antigens with glycosidically linked bridging arms. The attachment chemistry for various conjugates is described in one embodiment of an attachment scheme as shown in Example XI, columns 17-19.
U.S. Pat. Nos. 4,220,008 and 5,254,676 to Sabesan (1993) describe inhibitors for influenza virus. The inhibitors are heptasaccharide compounds with various side chains (see column 2). In one embodiment, R.sup.1 is (CH.sub.2).sub.n CONHR.sup.3 NHC(O)R.sup.4.
Hallren and Hindsgaul, J. Carbohydrate Chem. (1995) describes linkers for fucose. In one embodiment, fucose is linked to biotin using a spacer.
Probert et al., Carbohydrate Res. (1996) describes various glycan epitopes. Synthetic carbohydrate molecules with side chains are shown.
U.S. Pat. No. 4,255,566 to Carrico and Johnson (1981) describes flavin adenine dinucleotide derivatives.
Jacobson et al., J. Med. Chem. (1987) describes adenosine conjugates where the structure of interest is used as a linker to peptides.
U.S. Pat. No. 5,424,297 to Rubio et al. (1995) describes conjugates of dextran with adenosine. FIG. 1C shows a conjugate where the structure of interest is used as the linker.
Larionova et al., Biol. Chem. (1985) describes the conjugation of aprotinin with dextran derivatives of D-galactose. The structure of interest is shown in Scheme 2.
Pozsgay et al., J. Org. Che. (1997) describes the conjugation of kojidextrins (oligosaccharides) to proteins. Side chains containing the structure of interest are shown.
Klyashchitsky and Mitina, J. Chromatography (1981) shows the use of the structure of interest in making affinity adsorbents.
Inman and Barnett, J. Chromatography (1986) describes the functionalization of agarose. The structure of interest is shown in FIG. 1 as a spacer.
U.S. Pat. No. 4,966,607 to Shinoki and Ono (1990) describes starch derivatives containing side chains (some of which have the structure of interest) for conjugation to dyes.
V. Pavliak, P. Kovac, C. P. J. Glaudemans, Synthesis of Ligans Related to the O-Specific Antigen of type 1 Shigella Dysenteriae. 2. Stereoselective syntheses of a di-, tri-, and a tetrasaccharide fragment of Shigella dysenteriae type 1 O-antigen using 3,4,6-tri-O-acetyl-2-azido-2-deoxy-.alpha.-D-glucopyranosyl chloride as a glycosyl donor, Carbohydr. Res., 229 (1992) 103-116.
P. Kovac, K. J. Edgar, Synthesis of Ligands Related to the O-Specific Antigen of type 1 Shigella Dysenteriae. 3. Glycosylation of 4,6-O-substituted derivatives of methyl 2-acetamido-2-deoxy-.alpha.-D-glucopyranoside with glycosyl donors derived from mono- and oligosaccharides, J. Org. Chem., 57 (1992) 2455-2467.
P. Kovac, Synthesis of Ligands Related to the O-Specific Antigen of Shigella Dysenteriae type 1, 4. Enhanced stereoselectivity of .alpha.-D-galactosidation in the synthesis of the sequence .alpha.-D-gal-(1.fwdarw.3)-.alpha.-D-GlcNAc, allowing further extension of the chain at C-2', J. Carbohydr. Chem., 11 (1992) 999-1014.
P. Kovac, Di- and trisaccharide glycosyl donors for the synthesis of fragments of the O-specific antigen of Shigella dysenteriae type 1, Carbohydr. Res., 245 (1993) 219-231.
It is an object of the present invention to provide spacers for linking mono- oligo- or polysaccharides from which glycosyl donors can be made to amine-containing carriers, and a method of synthesis of such spacers.
It is a further object of the present invention to provide a coupling procedure in which an intermediate product can be isolated, purified and characterized prior to the coupling reaction.
It is yet a further object of the present invention to provide a heterobifunctional spacer or linking compound which can be used under mild chemical transformation conditions to carry out a coupling reaction.
A still further object of the present invention to provide a coupling procedure which does not adversely affect the structure or conformation of the materials which are coupled.
It is yet another object of the present invention to couple antigens based on natural, modified natural or synthetic mono- oligo- or poly-saccharides to an amine-containing carrier. It is also an object of the invention to couple molecules which are structurally related and/or antigenically similar to those mono- oligo- and poly-saccharides to a carrier material. Preferably, these mono- oligo- or poly-saccharides used in the invention are antigenically similar to known antigenic determinants.
It is yet another object of the present invention to provide mono- oligo- or poly-saccharide conjugates which are useful as vaccines.